Dendritic cells infected with recombinant adenoviral vector encoding mouse fibroblast activation protein‐α and human livin α exert an antitumor effect against Lewis lung carcinoma in mice

Abstract Background Fibroblast activation protein‐α (FAP) and livin α are considered as cancer‐associated fibroblasts (CAFs) and tumor‐specific targets, respectively, for immunogenic tumor vaccines. This study is designed to decipher the antitumor effect of double‐gene modified dendritic cells (DCs) on Lewis lung carcinoma (LLC). Methods By encoding mouse FAP cDNA and human livin α (i.e., hlivin α) cDNA into recombinant adenoviral vector (rAd), rAd‐FAP, rAd‐hlivin α, and rAd‐FAP/hlivin α were constructed, which were then transduced into mouse DCs. LLC‐bearinig mice were immunized with the infected DCs (5 × 105 cells/mouse), followed by calculation of tumor volume and survival rate. The identification of CAFs from mouse LLC as well as the determination on expressions of FAP and livin α, was accomplished by western blot. Cytotoxic T lymphocyte assay was harnessed to assess the effect of the infected DCs on inducing splenic lymphocytes to lyse CAFs. Results DCs were successfully transduced with rAd‐FAP/hlivin α in vitro. FAP was highly expressed in CAFs. CAFs were positive for α‐SMA and negative for CD45 and CD31. Livin α level was upregulated in mouse LLC. Immunization with rAd‐FAP/hlivin α‐transduced DCs suppressed LLC volume and improved the survival of tumor‐bearing mice. Immunization with rAd‐FAP/hlivin α‐transduced DCs enhanced the cytotoxic effect of splenic lymphocytes on LLC tumor‐derived CAFs. Conclusion Injection with rAd‐FAP/hlivin α‐transduced DCs promotes immune‐enhanced tumor microenvironment by decreasing CAFs and suppresses tumor growth in LLC mouse models.


| INTRODUCTION
Lung cancer is the second most common type of malignancy in the world, with which patients generally have a low 5-year survival rate and poor prognosis. 1][4][5] With the advance of biological therapy, there is a growing interest in the tumor-specific cytotoxicity of cytotoxic T lymphocytes (CTLs). 6Previously, increasing the number of CTLs and activating CTLs towards effector cells against cancer cells have been considered promising strategies for making efficient antitumor immune responses. 7It is believed that CTLs with CD8 + surface marker are primed by dendritic cells (DCs), CD4 + T cells, and natural killer cells. 8Among them, DCs play a central role in the immune response and are able to induce tumor-specific CTL response by professionally presenting antigens to naïve T lymphocytes, thereby activating CTL to destroy antigen-expressing cancer cells. 9However, cancer cells and stromal cells within the tumor microenvironment (TME) exert immunosuppressive properties that disturb the antitumor immunity of DCs. 10 Therefore, it is important to genetically modify DCs based on appropriate tumor targets for the development of effective tumor vaccines.
Fibroblast activation protein-α (FAP) is a membranebound serine protease and is specifically expressed on the surface of reactive cancer-associated fibroblasts (CAFs) that constitute a major stromal component of most solid tumors. 11Available evidence has demonstrated that FAP plays a critical role in the formation of TME, in which this cell surface protease can shape key features of CAFs through proteome and degradome alterations to suppress antitumor immunity and facilitate tumor growth and metastasis. 12In recent years, the depletion of FAP in CAFs by multiple methods, including cell-based vaccines has been reported to induce the immune system and inhibit tumor progression. 13,14ivin belongs to the family of antiapoptotic proteins and is poorly expressed in most normal tissues but overexpressed in most common human cancer cells. 15,16n view of the contributing role of livin in proliferation, antiapoptosis, and resistance of cancer cells as well as good prognosis, [17][18][19] it has been widely thought as a specific gene in tumor tissues and could be used as a promising target for tumor immunotherapy.
Previously, we confirmed that human livin α (i.e., hlivin α)-transduced DCs enhanced tumor-specific CTL response for killing Lewis lung carcinoma (LLC) cells as well as inhibited tumor growth in mice, yet this antitumor vaccination failed to realize tumor eradication, as the tumor-bearing mice eventually died from the tumors (Junping 20 ).In this study, we modified mouse bone marrow-derived DCs by infecting the cells with recombinant adenoviral vector (rAd) encoding transgenes to explore the immunogenicity of mouse FAP combined with human livin α on LLC in mice so as to improve the efficacy of LLC vaccine.

Experimental animals and ethics statement
Female C57BL/6 (H-2 b ) mice (6-8 weeks old) were reared in a pathogen-free environment with food and drinking water ad libitum.All experiments involving animals in this study had been granted by the Ethics Committee of Zhejiang Baiyue Biotech Co., Ltd. for Experimental Animals Welfare (Approval No. ZJBYLA-IACUC-20221104), and all procedures abided by the guidelines of the China Council on Animal Care and Use.

| Establishment of LLC mouse models and in vivo immunization
The cultured LLC cells were digested and collected in PBS at a concentration of 2.5 × 10 6 cells/mL.To establish LLC-bearing mice, 5 × 10 5 cells were subcutaneously injected into the right flank of each mouse. 14o investigate the antitumor activity of DC vaccine in vivo, LLC mouse models, whose tumor diameter reached 4-6 mm on the 8th day after LLC cell injection, were randomly divided into four groups (n = 10), and mice in each group were subjected to immunization with rAd-EGFP-transduced DCs (1#), rAd-hlivin α-transduced DCs (2#), rAd-FAP-transduced DCs (3#), and rAd-FAP/ hlivin α-transduced DCs (4#) (5 × 10 5 cells/mouse), respectively.Immunization was given by subcutaneous injection into the left flanks of mice for a total of three times every 3 days.Every 2 days after the first vaccination, the longest diameter (length) and shortest diameter (width) of mouse tumors were measured with a vernier caliper to calculate the tumor volume following the formula: V = length × width 2 × 0.52 (mm 3 ) (Junping 20 ).All mice were euthanized (50 mg/kg pentobarbital sodium, P-010, Sigma-Aldrich) when the tumor diameter reached 20 mm in vivo, with death time recorded accordingly for calculation of survival rate.The observation period for the survival rate of tumor-bearing mice lasted for 80 days.

| CAF isolation
The isolation of CAFs from implanted LLC was conducted as described previously. 21In brief, tumor tissues were excised from mice about 18 days after LLC cell injection and cut into 1-2 mm 3 pieces.10% FBScontaining DMEM was used to cover tissue pieces in T25 cell culture flasks at 37°C with 5% CO 2 , with medium replaced every 48 h.After 2 weeks, trypsinization (0.25% trypsase, C0201, Beyotime) was performed for 5 min, and cell pellets were allowed to grow adherently in the medium, followed by PBS washing.The purified CAFs were normally cultured and used for following assays at Passage 4-5.

| CTL assay
Splenic lymphocytes were obtained from mice on Day 7 after receiving the last dose of DCs vaccination and then were cultured in complete medium (CM-M153, Procell) at 37°C with 5% CO 2 for 90 min.CAFs pretreated with Mitomycin C (25 mg/L, M5791, Abmole) were used to stimulate splenic lymphocytes in the medium containing IL-2 (M19999, Abmole).After 5 days of incubation, the stimulated lymphocytes were harvested as effector cells and then added into 96-well plates with CAFs (target cells) at a ratio of 20:1, 40:1, or 60:1.The cytotoxic effect of effector cells on target cells was examined by Cytotox96 Non-Radioactive Cytotoxicity Assay Kit (G1780, Promega) according to the manufacturer's protocol.A microplate reader (Multiskan FC, Thermo Fisher) was employed to detect cell absorbance at 450 nm, followed by cytotoxicity calculation. 22

| Statistical analysis
Measurement data were obtainable from three repeated experiments and shown as mean ± standard deviation.Comparisons among different groups were analyzed using one-way analysis of variance, and comparison between two groups in Figure 2 was analyzed using independent samples t-test.Survival was analyzed by Kaplan-Meier survival curve and log-rank test.Graph-Pad Prism 8.0 (GraphPad Software Inc.) was utilized for statistical analysis, and statistical significance was set at p < .05.

| DCs were successfully transduced with rAd-FAP/hlivin α in vitro
After mouse DCs were infected with rAd carrying FAP or/and hlivin α, western blot was performed to analyze transduction efficiency.Compared with that in rAd-EGFP-infected cells, an increasing trend towards the expression of FAP was observed in rAd-FAP-infected cells, and an elevated tendency towards the expression of livin α was viewed in rAd-hlivin α-infected cells (Figure 1A-C, p < .001).In rAd-FAP/hlivin α-infected cells, FAP and livin α expressions were both increased when compared with those in rAd-EGFP-infected cells and rAd-hlivin α-infected cells (Figure 1A-C, p < .001).Collectively, these findings indicated the successful transduction of DCs with rAd-FAP and rAd-livin α in vitro.

| FAP was highly expressed in CAFs from mouse LLC
Next, we isolated CAFs from LLC tumor-bearing mice to detect epithelial cell adhesion molecules (CD45 and CD31) and α-SMA.The results of western blot verified that CAFs were positive for α-SMA protein (Figure 2A, p < .001)and negative for CD45 and CD31 proteins.Compared with that in mouse LLC, FAP level was found to be significantly upregulated in CAFs (Figure 2B, p < .001).

| Livin α level was upregulated in mouse LLC
It has previously confirmed that livin α is specifically expressed in multiple tumor cells, and its downregulation shows an antitumor effect. 23Moreover, the expression of livin α was demonstrated to be increased in mouse LLC tissues, as compared with that in paracancerous tissues (Figure 2C, p < .01).

| Immunization with rAd-FAP/ hlivin α-transduced DCs suppressed LLC volume and improved the survival of tumor-bearing mice
In the following therapeutic efficacy study, the volume of LLC in mice injected either with rAd-hlivin α-transduced DCs or rAd-FAP-transduced DCs was decreased (Figure 3A, p < .001),and this suppressing effect was strengthened in the presence of rAd-FAP/hlivin α-transduced DCs (Figure 3A,B, p < .05).Moreover, tumor-bearing mice who received immunization with rAd-FAP/hlivin α-transduced DCs showed higher survival rate compared with those receiving immunization with rAd-hlivin α-transduced DCs or rAd-FAP-transduced DCs alone (Figure 3C, Table 1).

| Immunization with rAd-FAP/ hlivin α-transduced DCs enhanced the cytotoxic effect of splenic lymphocytes on LLC-derived CAFs
With regard to the contributing role of CAFs in the formation of tumor microenvironment, we investigated whether rAd-FAP/hlivin α-transduced DCs could enhance the cytotoxic effect of splenic lymphocytes on CAFs in vitro.As shown in Figure 3D, splenic lymphocytes from mice immunized with rAd-FAP-transduced DCs exhibited an increased cytotoxicity to CAFs (p < .001),whereas splenic lymphocytes from mice immunized with rAd-hlivin α-transduced DCs showed no significant cytotoxic effect on CAF.Of note, splenic lymphocytes from mice immunized with rAd-FAP/hlivin α-transduced DCs exerted stronger cytotoxicity against CAFs than those from mice immunized with rAd-hlivin α-transduced DCs or rAd-FAP-transduced DCs alone (Figure 3D, p < .05).

| DISCUSSION
Although targeted therapy and immune checkpoint inhibitors have improved the treatment landscape for patients with lung cancer in recent years, the clinical efficacy is still unsatisfactory. 24Hence, the tremendous endeavor has been dedicated to finding novel and effective therapy with low toxicity.In this study, twoantigen-loaded DC vaccines are successfully built through infection with rAd encoding mouse FAP and human livin α, which have been reported as an efficient tool for transferring genes into DCs. 19Based on the results of the therapeutic efficacy study in vivo, we revealed for the first time that rAd-FAP/hlivin α-transduced DCs suppressed tumor volume and improved the survival of LLC mouse models, and these effects were more pronounced than those of rAd-hlivin α-transduced DCs or rAd-FAP-transduced DCs.Furthermore, we demonstrated that rAd-FAP/ hlivin α-transduced DCs showed a more significant effect on inducing CTL responses to kill CAFs than rAd-FAP DCs.
As the most potent antigen-presenting cells, DCs have always been a major interest in the development of tumor vaccines. 9,25Compared with antigen-adjuvant vaccines, a previous study has suggested that antigenloaded DC vaccines exhibit a stronger effect on inducing T-cell immune responses against mouse tumors. 26ecently, tumor-associated antigen-pulsed DC vaccines for cancer immunotherapy have been studied in clinical trials and reported to obtain a good therapeutic effect, 27,28 yet their effectiveness and sustained antitumor immunity remain controversial because of the adaptive immunomodulatory mechanisms of TME and the mutational nature of the cancer cell genome.Existing studies have documented that livin protein is overexpressed in many lung cancer cell lines and primary lung cancers and that many patients with lung cancer have anti-livin antibodies, as well as anti-livin cellular immune responses, [29][30][31] hinting the potential of livin as a target for the immunotherapy of lung cancer.In this study, we also found that LLC excised from mice presented overexpression of livin α.
With an in-depth understanding of tumorigenesis, researchers have discovered that CAFs are closely associated with the development of immunosuppression by interacting with immune cells in TME and have emerged as a novel interstitial target for tumor immunotherapies designed to complement cancer celltargeted therapies. 32,33CAFs, which are characterized as α-SMA-marked myofibroblasts, show a more stable genome than cancer cells, 34 suggesting that they have a low risk of antigen loss and treatment tolerance in the immunotherapy of solid tumors.It is widely known that FAP is highly expressed in tumor stroma from patients with lung cancer, and FAP overexpression can facilitate the proliferation of CAFs as well as cancer cells in vitro and in vivo. 35Compared with FAP-negative CAFs, FAPpositive CAFs have been found to facilitate tumor growth of gastric cancer in vivo as well as inhibit T-cell activation and infiltration, which could be responsible for the failure of antitumor immunity. 36In the study of breast cancer, Xia et al. have indicated that FAP-based vaccines can enhance the specific immune response for eliminating CAFs, which is an attractive way to overcome immunosuppression in combination with antitumor agents. 37In LLC mouse models, we confirmed that FAP was preferentially expressed in CAFs in comparison with that in solid tumors.Similar to the previous study, 14 we observed a significant antitumor effect of rAd-FAPtransduced DCs on LLC by reducing tumor volume, increasing survival rate, and enhancing CAF-specific cytotoxicity, and intriguingly this effect was further strengthened in the presence of livin α-targeted vaccination.Taken together, it is indicated that double-genemodified DC vaccines are more effective in combating LLC than single-gene-modified DC vaccines.
In conclusion, the present study suggests that injection with rAd-FAP/hlivin α-transduced DCs promotes immune-enhanced TEM by killing CAFs and suppresses tumor growth, thereby prolonging mouse survival, which is an advancement in the field of tumor immunology.Also, our current findings support the advantage of DCs as efficient gene vehicles in the development of tumor vaccines.As FAP and livin α are specifically expressed in CAFs and LLC cells, respectively, immunotherapy based on the combination of the two may have a huge potential to overcome TEM-caused immunosuppression and deliver a one-two punch to LLC.

F I G U R E 2
Identification of LLC tumor-isolated CAFs and determination on expressions of FAP and livin α in LLC.(A-C) Western blot was performed to measure protein expressions of CD45, CD31, α-SMA, and FAP in CAFs isolated from LLC-bearing mice as well as the protein expression of livin α in mouse LLC tumors.GAPDH was used as the loading control.+++ p < .001,versus lysate; ^^^p < .001,versus tumor; ## p < .001,versus Para.CAFs, cancer-associated fibroblasts; LLC, Lewis lung carcinoma; Para, paracancerous tissues.

cn,
the number of an individual still alive at time T. e S (t), the probability of an individual surviving beyond time T.
a T, survival time.b d, the number of event occurrences.